Identification, characterization, and method of production of a novel microbial lipase

ABSTRACT

A novel lipase from a newly-discovered strain of Pseudomonas alcaligenes microorganism having (i) an optimum pH for activity of about 10±0.5; (ii) an optimum temperature for activity of about 45° to 55° C.; (iii) an optimum pH for stability of about 7.0±0.5; (iv) a molecular weight as measured by gel permeation chromotagraphy of about 8.8×10 4  ; and (v) chemical stability for at least 30 days in the presence of the surfactants. Also claimed is a biologically pure culture of the microorganism, and a method for the production of the lipase.

This invention herein described relates generally to a biologically-pureculture of a newly-isolated microorganism, a novel microbial lipaseproduced by this microorganism and its characterization, and methodologyfor its production. More specifically, the invention relates to theisolation from nature of a new strain of Pseudomonas alcaligenes capableof producing a novel lipase under laboratory conditions. Characteristicsof this lipase, and methods for its production, are described.

By way of background, the literature is replete with references to theuse of microoganisms for the production of commercially importantproducts. Although microscopic unicellular algae and protozoa serve asthe source of some commercial products, the most common microorganismsfor such purposes are certain types of bacteria and fungi. Commercialproducts from these microorganisms range from gaseous by-products, toantibiotics, to food additives, and various extracellular orintracellular products such as enzymes and industrial chemicals.Bacterial or fungal isolates are commonly obtained from nature, broughtinto axenic culture, and used as active ingredients for variousdegradation treatments or to produce pharmaceuticals or other desirablechemicals.

Various types of microbial enzymes have been reported in the technicaland patent literature. For example, the production of proteolytic enzymepreparations from strains of Bacillus alcalophilus is described in U.S.Pat. No. 3,674,643. Microbial lipases have been widely reported in thetechnical literature. For example, lipases have been described inrepresentatives of the following microbial genera: Rhizopus, Fusarium,Helminthosporium, Mucor, Candida, Phycomyces, Aspergillus, Sclerotinia,Pseudomonas, Pneumococcus, Staphylococcus, Mycobacterium, Mycotorula,Chromobacterium, Achromobacterium, Brevibacterium, Corynebacterium,Alcaligenes, and Acinetobacterium. The lipases found among thesemicrobial genera and other microorganisms are quite diverse andtypically vary from one another in physical, chemical, and biologicalproperties.

The bacterial genus Pseudomonas consists of a group of aerobic, gramnegative, non-spore-forming, rod-shaped bacteria. The organism P.alcaligenes, a strain of which is the subject of this invention, is anaerobic pseudomonad which along with P. pseudoalcaligenes is included inwhat is known as the "Pseudomonas alcaligenes" group [Ralston-Barrett,E., N. J. Palleroni, and M. Doudoroff, Inter. J. System. Bacteriol,26(4): 421-426 (1976)].

The patent literature is replete with disclosures relating to microbiallipolytic enzymes and their various uses. Examples of such patents andpatent applications are: U.S. Pat. Nos. 3,511,753; 4,283,494; and4,318,818; European Patent Application Nos. 0 214 761, 0 130 069, and 0130 064; and Patent Cooperation Treaty International Publication No. WO87/00859. Other types of bacterial enzymes, such as proteases are alsodisclosed in the patent literature, as illustrated by U.S. Pat. No.3,674,643. All of these publications are incorporated herein byreference in their entirety.

One of the major commercial uses of microbial lipases is as cleaningcompositions, including detergents, to break down lipids. Although alarge number of lipolytic enzymes are known in microorganisms, not allsuch enzymes are suitable for commercial utilization. Such factors as pHrange, tolerance of emulsifiers and surfactants, temperature tolerance,storage capability and the like, are important considerations in theselection and development of a commercially useful product.

In one aspect the present invention relates to a bacterium whichproduces a novel active lipase. This bacterium, having been isolatedfrom nature and placed in axenic culture, is identified as a strain ofPseudomonas alcaligenes.

In another aspect, the present invention relates to a lipasecharacterized by having (i) an optimum pH for activitiy of about 10±0.5;(ii) an optimum temperature for activity of about 45° to 55° C.; (iii)an optimum pH for stability of about 7.0±0.5; (iv) a molecular weight asmeasured by gel permeation chromotagraphy of about 8.8×10⁴ ; and (v)chemical stability for at least 30 days in the presence of an aqueouscalcium chloride solution of surfactant(s), particularly the surfactantspolyoxyethylene (23) lauryl ether (commercially available as BRIJ® 35, aproduct of Atlas Chemical), and sodium trideceth-7-carboxylate(commercially available as SANDOPAN® DTC, a product of Sandoz).

In yet another aspect, the present invention relates to a method forproducing the microbial lipase SD2 comprising the steps of:

(a) aerobically cultivating the microorganism P. alcaligenes strain SD2,ATCC 53877 under conditions suitable for the formation of said lipase ina nutrient culture medium containing assimilable sources of carbon,nitrogen, and inorganic minerals at a pH of about 5 to 12 and atemperature of about 5° to 40° C.;

(b) incubating the culture for a period sufficient to provide productionof lipase, for example 18 to 24 hours; and

(c) recovering said lipase from said nutrient culture medium;

for the laboratory growth of this bacterium and production of a novellipase.

FIG. 1 is a graphic representation of the effect of pH on the enzymaticactivity of a preferred lipase obtained from Pseudomonas alcaligenesstrain SD2, a preferred microorganism of the invention.

FIG. 2 is a graphic representation of the effect of temperature on theenzymatic activity of a preferred lipase of the invention produced fromP. alcaligenes strain SD2, as compared to a lipase produced from P.alcaligenes strain ATCC 14909.

The present inventor has now isolated a biologically-pure culture of apreviously undescribed strain of Pseudomonas alcaligenes strain SD2. Theorganism is a natural isolate and has been deposited with the AmericanType Culture Collection (ATCC), having been assigned the accessionnumber ATCC 53877. This novel strain SD2 was found to produce a novellipase.

The microorganism of the invention, P. alcaligenes strain SD2 wasisolated from a shower drain by direct isolation on aTryptone-Soytone-Olive oil isolation medium. The isolation mediumemployed is more fully described in Table I below.

                  TABLE I                                                         ______________________________________                                        Isolation Medium                                                                                Percent by Weight                                           ______________________________________                                        Ammonium sulfate    0.5                                                       Potassium phosphate, dibasic                                                                      0.05                                                      Magnesium sulfate, heptahydrate                                                                   0.025                                                     Tryptone (Difco)    1.7                                                       Soytone (Difco)     0.3                                                       Olive oil           1.0                                                       Rhodamine B         0.001                                                     Agar                1.5                                                       ______________________________________                                    

The Rhodamine B dye in the isolation medium causes lipase-producingbacterial colonies to fluoresce an orange color when irradiated withlong wavelength ultraviolet light (Kouker, G. and K.-E. Jaeger, 1987,Appl. Environ. Microbiol., 53: 211-3). This fluorescence permits theeasy identification of lipase-producers. Colonies so identified werepurified by restreaking onto similar media. Stock cultures weremaintained in Difco TSA slants.

The bacterial isolate of the invention was identified using standardtaxonomic procedures from Bergey's Manual of Systematic Bacteriology(Williams & Wilkins, Baltimore, 1984). The results of applicablephysiological characterization tests of P. alcaligenes strain SD2 arepresented in Table II and compared with characteristics of P.alcaligenes and P. pseudoalcaligenes published in Bergey's Manual.

                  TABLE II                                                        ______________________________________                                        Substrate Utilization of P. alcaligenes                                       Strain SD2, P. alcaligenes, and P. pseudoalicaligenes                                     Strain*                                                                  SD2    P. alcaligenes                                                                           P. pseudoalcaligenes                                 ______________________________________                                        Fructose -        -          +                                                L-aspartate                                                                            +        -          -                                                L-glutamate                                                                            -        +          +                                                D-gluconate                                                                            -        -          d                                                L-Histidine                                                                            -        d          d                                                Ethanolamine                                                                           -        -          +                                                n-Butanol                                                                              -        d          +                                                Isobutanol                                                                             +        d          -                                                Citrate  -        d          d                                                Betaine  -        -          +                                                Glycerol -        -          d                                                Sorbitol -        -          d                                                Itaconate                                                                              -        -          d                                                ______________________________________                                         Abbreviation:                                                                 d (11-80 percent of strain positive);                                         + (strain was able to utilize the indicated chemical for growth);             - (strain did not utilize the chemical for growth).                           *Data for P. alcaligenes and P. pseudoalcaligenes are from Bergey's Manua     of Systematic Bacteriology (Williams & Wilkins [Baltimore, 1984]).            Compounds utilized by all strains include: DLlactate, succinate, fumarate     acetate, Larginine, caprate, and Lmalate.                                     Compounds not utilized by any strain include: Dglucose, Larabinose,           Dmannose, Dmannitol, α-Lrhamnose, D (+)galactose, D (-)ribose,          minositol, Lthreonine, mtartrate, adipate, phenylacetate, nicotinate,         sebacate, suberate, benzoate, and pimelate.                              

This table illustrates nutritional capabilities of the indicated strainsand further illustrates their differences.

Several lipase-producing strains of P. pseudoalcaligenes are disclosedin International Publication No. WO 87/00859 published under the PatentCooperation Treaty. Table III presents certain morphological andphysiological characteristics of P. alcaligenes strain SD2, as comparedto the characteristics of four strains of P. pseudoalcaligenes disclosedin International Publication No. WO 87/00859. Differences between theSD2 strain of the present invention and the other strains are readilyapparent. For example, SD2 utilized L-aspartate, while the two otherPseudomonas species did not.

                  TABLE III                                                       ______________________________________                                        Characteristics of P. alcaligenes Strain SD2 and                              Selected Lipase-Producing Strains of P. pseudoalcaligenes.                    (The CBS Strain Accession Numbers Correspond to Those                         Referenced in International Publication No. WO 87/00859)                              Strain                                                                        of      Comparison Strains                                                      Invention CBS     CBS   CBS    CBS                                  Characteristic                                                                          SD2       467.85  468.85                                                                              471.85 473.85                               ______________________________________                                        Cell shape                                                                              rod       rod     rod   rod    rod                                  Motility  +         +       +     +      +                                    Spores    -         -       -     -      -                                    Gram strain                                                                             -         -       -     -      -                                    Oxidase   +         +       +     +      +                                    Anaerobic -         -       -     -      -                                    glucose                                                                       Aerobic   -         -       -     -      -                                    glucose                                                                       Aerobic   -         -       -     -      -                                    maltose                                                                       Aerobic   -         -       -     -      -                                    sucrose                                                                       Aerobic   -         -       -     -      +                                    D-xylose                                                                      Arginine  +         +       +     -      +                                    dihydrolase                                                                   Gelatin   -         -       -     -      -                                    hydrolysis                                                                    Starch    -         -       -     -      -                                    hydrolysis                                                                    NO.sup.-.sub.3 →NO.sup.-.sub.2                                                   +         +       +     +      +                                    NO.sup.-.sub.2 →N.sub.2                                                          +         -       -     -      -                                    Citrate   -         +       +     +      +                                    Utilization                                                                   Catalase  +         +       +     +      +                                    Growth at +         +       +     +      +                                    41° C.                                                                 ______________________________________                                    

Strain SD2 of the present invention can be grown in various types ofculture media under conditions suitable for growth of pseudomonads.Typically, such media contain assimilable sources of carbon, nitrogen,and various inorganic mineral nutrients. By way of illustration, P.alcaligenes strain SD2 was grown in Tryptone Medium having theformulation as shown in Table IV.

                  TABLE IV                                                        ______________________________________                                        Culture Medium                                                                Ingredient          Percent by Weight                                         ______________________________________                                        Ammonium sulfate    0.5                                                       Potassium phosphate, dibasic                                                                      0.05                                                      Magnesium sulfate, heptahydrate                                                                   0.025                                                     Tryptone (Difco)    2.0                                                       BRIJ ® 58       1.0 mM                                                    ______________________________________                                    

The lipase of the invention is found in culture media, preferably liquidmedia, containing P. alcaligenes strain SD2. Quantities of this enzymecan be obtained by culturing P. alcaligenes strain SD2 in liquid cultureand under culture conditions suitable for growth of organisms of thistype. For example, an actively growing aliquot of P. alcaligenes strainSD2 is suitably used as an innoculum and introduced into Erlenmeyerflasks containing Tryptone medium (C.F. Table IV). Cultures areincubated with shaking for about 16 to 18 hours at a temperature ofabout 30° C. Following this culture growth period, the bacterial cellsare removed by centrifugation or filtration or other suitabletechniques. The lipase which is found in the resultant clarified cultureliquor is then generally concentrated prior to use. Several methods maybe used to concentrate this enzyme, including ultrafiltration asdiscussed in Example 1.

It is desirable that lipases intended for commercial utilization bestable in the presence of various surfactants commonly found in cleaningproduct formulations. Advantageously, the lipase of P. alcaligenesstrain SD2 was found to be functional in the presence of commercialsurfactants such as dodecylbenzene sulfonate and fatty alcoholethoxylates. In addition, the inclusion of the non-ionic surfactantBRIJ® 35 [polyoxyethylene (23) lauryl ether] in liquid growth mediumcontaining P. alcaligenes strain SD2 at a 1-10 mM concentration,preferably 1 mM, increased the yield of the lipase by a factor oftwo-fold or more in contrast to control cultures without thissurfactant.

Regarding the stability of the lipase produced by P. alcaligenes strainSD2, this enzyme loses activity during storage at a rate that isdirectly proportional to temperature. For example, during acceleratedaging tests conducted at a temperature of 37° C. and a pH of 7.0, thelipase of the invention demonstrated a half-life of about 5 days in theabsence of surfactants. The addition of calcium, in the form of CaCl₂,stabilized the SD2 lipase and increased its half-life to over 80 days atsuitable CaCl₂ concentrations. The concentration of CaCl₂ required toenhance such enzyme longevity is related to the particular lipaseformulation. For example, in simple buffered enzyme solutions lackingsurfactants, where the buffer is, for example, 50 mM BES [N, N-bis(2-hydroxyethyl)-2-amino-ethanesulfonic acid] at pH 7.0, the addition of5 mM CaCl₂, preferably 10 mM, is sufficient. The optimum concentrationof CaCl₂ in the presence of preferred surfactants is about 25 mM ormore. In formulations of the lipase of P. alcaligenes strain SD2,various surfactants can be used in view of this lipase's stability inthe presence of surfactants as illustrated in Table VI below. Examplesof preferred surfactants include the nonionic surfactant BRIJ® 35[polyoxyethylene (23) lauryl ether] and the anionic surfactantSANDOPRON® DTC gel. Preferred nonionic surfactants are those having ahydrophobic end containing 12-16 carbon units, and a polyoxyethylenechain size of about 20-23 ethylene oxide units. In general, anionicsurfactants of the carboxylated type are preferred and are mostcompatible with the novel lipase of P. alcaligenes strain SD2.

The following examples further serve to illustrate the invention, butare not intended to be limitative thereof.

EXAMPLE 1 Preparation of Lipase From Pseudomonas alcaligenes Strain SD2

The microorganism of the invention, P. alcaligenes SD2, was convenientlygrown in the culture medium previously presented in Table IV.

A 50 mL starter culture of P. alcaligenes SD2 in a 250 mL Erlenmeyerflask was grown for about 16 hours at a temperature of 30° C. at 175 rpmon a gyratory shaker. This starter culture was then used to inoculate 8liters of culture medium which was in turn placed in 4 and 6 L flutedErlenmeyer flasks such that no individual flask contained more than 25percent flask capacity as liquid. The culture flasks thus prepared wereincubated for 24 hours at a temperature of 30° C. with gyratory shakingat 150 rpm.

Following the culture period, the lipase of the invention is harvestedand concentrated by first removing the bacterial cells from the 8 litersof liquid culture by tangential flow filtration using Pharmacia 10⁶(NMWC) Omega membrane cassettes. The resultant cell-free filtrate wasthen concentrated by tangential flow ultrafiltration using Pharmacia30,000 (NMWC) Omega membrane cassettes. Thereafter, the concentrate wasdiafiltered at 3° C. with about 10 volumes of 50 mM BES, pH 7.0,supplemented with 10 mM CaCl₂ in order to eliminate all low molecularweight contaminants (those with molecular weights less than or equal to30,000), and to change the lipase solvent to one with buffer andstabilizing CaCl₂. The yields of enzyme from three separate batchcultures are presented in Table V.

                  TABLE V                                                         ______________________________________                                        Yields of Lipase Produced by Cultures                                         of P. alcaligenes Strain SD2                                                  Batch No.      Units/mL.sup.(1)                                                                        Total Units                                          ______________________________________                                        20             39.15     10,571                                               21             34.69     7,840                                                22             37.41     6,172                                                ______________________________________                                         .sup.(1) 1 Unit is the amount of lipase production from olive oil 1μ       equivalent fatty acid per minute at 37° C. at pH 10.              

EXAMPLE 2 Determination of Characteristics of the Lipase P. alcaligenesStrain SD2: Molecular Weight, and the Effects of Temperature and pH onLipolytic Activity

Quantities of the lipase of P. alcaligenes strain SD2 were obtained byculturing of the organism in the medium of Table IV, removing thebacterial cells by filtration, concentrating the enzyme byultrafiltration as already described. Lipolytic activity was assayedusing the following standard composition: (i) 2.5 mL substrate [10percent (w/v) olive oil in 10 percent (w/v) gum arabic]; (ii) 2.0 mLbuffer [1.0M CHES (2[N-cyclohexylamino]-ethane sulfonic acid), pH 10.0];(iii) enzyme; and (iv) distilled water added to a final volume of 6.0mL. Enzymatic assays were conducted at a temperature of 37° C. The fattyacids formed during the hydrolytic enzymatic reaction were extractedwith an organic solvent and titrated following the procedure describedin U.S. Pat. No. 4,283,494.

A quantity of the lipase of the invention was used to determine itsmolecular weight. The molecular weight of the lipase of P. alcaligeneswas found to be about 88,000 using gel filtration chromatography andcomparing the retention time of the lipase with molecular weightcalibration standards.

Using the standard procedure for determination of lipolytic activity asdescribed above, the effects of pH as well as temperature on theactivity of the lipase of the invention were determined. Results ofthese experiments are presented in FIGS. 1 and 2.

FIG. 1 shows the effect of pH on activity of the P. alcaligenes strainSD2 lipase. It can be seen that this lipase is active in the pH rangefrom less than pH 6.0 to over pH 11.0, and has an optimum pH of 10.0(c.f. FIG. 1). FIG. 2 shows the effect of temperature on activity of theP. alcaligenes strain SD2 lipase. Results of these experiments show thatthe lipase of the invention is active from a temperature of less than25° C. to over 50° C. and has a preferred temperature for optimumactivity of about 50° C. (c.f. FIG. 2).

EXAMPLE 3 Comparison of Characteristics of the Lipase of P. alcaligenesStrain SD2 With Other Selected Lipases

The lipase of P. alcaligenes was contrasted with lipases known to beproduced by the type strain of Pseudomonas alcaligenes (American TypeCulture Collection No. 14909), the Alcaligenes sp. of U.S. Pat. No.4,283,494 (American Type Culture Collection No. 31372) and the two NovoLipolase™. Samples of these enzymes were obtained by culturing therespective source organisms and extracting the enzyme from the culturemedia as described in Example 2 above. Stability of these enzymes in thesurfactants BRIJ® 35 and SANDOPAN® DTC was determined. In addition tothe determination of surfactant stability, the following characteristicsof the four microbial lipase were evaluated: optimum pH for enzymaticactivity; optimum pH for enzyme stability; optimum temperature forenzymatic activity; and molecular weight. Results of this comparison andassociated experiments are presented in Table VI.

                                      TABLE VI                                    __________________________________________________________________________    Lipase Characteristics                                                                               Strain                                                                  ATCC  ATCC   NOVO                                            Characteristic                                                                           SD2   14909.sup.(1)                                                                       31371.sup.(2)                                                                        Lipolase ™                                   __________________________________________________________________________    pH optimum 10    10     9     11                                              (activity)                                                                    pH optimum  7    --    --     --                                              (stability)                                                                   Temperature °C.                                                                   45-55 45-55 40-48  30-40                                           optimum (activity)                                                            Molecular  8.8 × 10.sup.4                                                                8.8 × 10.sup.4                                                                30-40 × 10.sup.4                                                               --                                              Weight                                                                        Surfactant Stability (mean half-life in days):                                BRIJ ® 35, 10%.sup.(3)                                                               60     5     --    11                                              SANDOPAN ® DTC                                                                       28     5    --     11                                              20%.sup.(3)                                                                   __________________________________________________________________________     .sup.(1) Pseudomonas alcaligenes type strain                                  .sup.(2) Alcaligenes sp. (ref. U.S. Pat. No. 4,283,494)                       .sup.(3) 25 mM CaCl.sub.2 added as stabilizer                            

It can be seen that the lipase of invention produced by P. alcaligenesstrain SD2 is novel and differentiable from other known lipases. The SD2lipase differs from the NOVO Lipolase™ and the lipase of Alcaligenes sp.(ATCC No. 31371) with respect to optimum pH for activity, optimumtemperature for activity, surfactant stability, and molecular weight(with respect to ATCC No. 31372; the molecular weight of NOVO Lipolase™is not known). The lipase of P. alcaligenes strain SD2 is comparable tothe lipases of the P. alcaligenes type strain (ATCC No. 14909) withrespect to all characteristics evaluated with the notable exception ofsurfactant stability. The lipase of the invention produced by P.alcaligenes strain SD2 shows exceptionally good stability in certainsurfactants tested. The stability of the SD2 lipase in surfactants isunique among the microbial lipases tested.

What is claimed is:
 1. A biologically-pure culture of the microorganismsPseudomonas alcaligenes strain SD2 having all of the identifyingcharacteristics of ATCC 53877 and derivatives and mutants thereof.
 2. Abiologically pure culture of the microorganisms Pseudomonas alcaligenesstrain SD2 according to claim 1, said culture being capable of producingthe lipase SD2 upon aerobic cultivation in an aqueous nutrient mediumcontaining assimilable sources of carbon, nitrogen, and inorganicsubstances, which lipase is characterized by having (i) an optimum pHfor activity of about 10±0.5; (ii) an optimum temperature for activityof about 45° to 55° C.; (iii) an optimum pH for stability of about7.0±0.5; (iv) a molecular weight as measured by gel permeationchromatography of about 8.8×10⁴ ; and (v) chemical stability for atleast a 60 day mean half-life in the presence of a 10 percent solutionof polyoxyethylene (23) lauryl ether in 25 millimolar aqueous calciumchloride.
 3. A biologically pure culture of the microorganism P.alcaligenes strain SD2, ATCC 53877 according to claim 2 capable ofproducing the lipase SD2 in recoverable amounts.
 4. An aerobic cultureof the microorganism P. alcaligenes strain SD2, ATCC 53877 whichproduces recoverable amounts of the lipase SD2.